ISPpu10 is a structure-gated bridge RNA recombinase that drives safe, large-scale genomic plasticity
IS110 elements employ bridge RNA to direct target site recognition, yet whether additional layers of specificity exist beyond RNA-DNA base pairing is unknown. Here we identified ISPpu10, an IS110 element in Pseudomonas putida that is associated with >100-kb segmental amplifications during adaptive laboratory evolution. All insertions mapped to multiple identical copies of a single conserved intergenic sequence harboring a GC-rich 5' extension that forms a stem-loop. Systematic mismatch and compensatory substitution scanning revealed a sequence-structure dual-match logic: base pairing between the bridge RNA and target DNA serves as an RNA-guided match, whereas the target-encoded stem-loop provides an additional specificity determinant. Transplanting this stem-loop into Escherichia coli targets improved on-target integration and reduced genome-wide off-target insertion, establishing the stem-loop as a portable structural gate. These findings reveal a structural recognition layer beyond base-pairing, suggesting how bacteria reconcile transposition with genome integrity and offering a strategy for precise genome editing.